Endonuclease

ABSTRACT

The present invention relates to a site-specific endonuclease which recognizes a specific nucleotide sequence, to a gene coding for the endonuclease, to a recombinant vector containing the gene, to a transformant containing the vector, and to a process for producing the endonuclease.

FIELD OF THE INVENTION

The present invention relates to a site-specific endonuclease whichrecognizes a specific nucleotide sequence, to a gene coding for theendonuclease, to a recombinant vector containing the gene, to atransformant containing the vector, and to a process for producing theendonuclease.

BACKGROUND OF THE INVENTION

Endonuclease is a nuclease (nucleic acid degrading enzyme) whichhydrolyzes the phosphodiester bond of a polynucleotide chain.Endonuclease recognizes and binds to a specific nucleotide sequencealong DNA molecules, whereby molecules within the recognition sequenceis cut. Endonuclease is a requisite enzyme for today's advanced geneengineering techniques for cloning and analyzing genes.

A site-specific endonuclease Endo.SceI (hereinafter, also referred to as“SceI”) from an eucaryotic microorganism (e.g., yeast) is known to be aheterodimer having subunits of 75 kDa and 50 kDa. The subunits of SceIas well as genes encoding the subunits have been cloned, and thenucleotide sequences thereof have been determined (for 75 kDa subunit,see Morishima, N. et al., J. Biol. Chem. 265, 15189-15197 (1990) and for50 kDa subunit, see JP-B-7-77556).

In order to widely utilize the above-described endonuclease forartificially modifying a biochemical agent, a gene or the like, theendonuclease needs to be mass-produced with a gene expression system.The endonuclease does not function unless it recognizes a specificnucleotide sequence, i.e., the endonuclease needs to be specific to thenucleotide sequence to be recognized.

The 50 kDa subunit of the above-described endonuclease SceI is encodedby mitochondrial genomes of yeast (Saccharomyces cerevisiae). A gene ofa mitochondrial genome of yeast contains codons unique to mitochondriawhich are different from amino acid codons (universal codons) used ingene expression systems from organisms generally used for massexpression of protein (E.coli, baculovirus, yeast, etc.). If this geneof the mitochondrial genome is directly used, the protein expressionsystem hardly produces a protein of an original amino acid sequence. Forexample, while TGA is a stop codon as a universal codon, it is adifferent codon coding for other amino acid (Trp) in mitochondria. Agene may be normally expressed in mitochondria but expression of thesame gene may not result in a complete protein in a general expressionsystem such as E.coli due to incomplete translation caused by the stopcodon.

SUMMARY OF THE INVENTION

The present invention aims at providing a site-specific endonucleasewhich recognizes a specific nucleotide sequence, to a gene coding forthe endonuclease, to a recombinant vector containing the gene, to atransformant containing the vector, and to a process for producing theendonuclease.

The present inventors have gone through intensive studies to solve theabove-described problems. As a result, they succeeded in producing amodified endonuclease capable of recognizing and cleaving a specificnucleotide sequence by substituting, in a gene encoding an amino acidsequence of the smaller subunit of an endonuclease from yeast, codonsunique to mitochondria with universal codons, and in mass-expressing theendonuclease, whereby the present invention was accomplished.

Accordingly, the present invention relates to an endonuclease capable ofrecognizing the nucleotide sequence: GCCCAGACATATCCCTGAATGATACC.

Further, the present invention relates to a recombinant protein ofeither (a) or (b):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:3; or

(b) a protein having an endonuclease activity for recognizing thenucleotide sequence: GCCCAGACATATCCCTGAATGATACC, the protein comprisingat least one deletion, substitution or addition of amino acid in theamino acid sequence represented by SEQ ID NO:3.

Moreover, the present invention relates to a gene encoding therecombinant protein of either (a) or (b):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:3; or

(b) a protein having an endonuclease activity for recognizing thenucleotide sequence: GCCCAGACATATCCCTGAATGATACC, the protein comprisingat least one deletion, substitution or addition of amino acid in theamino acid sequence represented by SEQ ID NO:3.

In addition, the present invention relates to a gene containing DNA ofeither (c) or (d):

(c) DNA comprising the nucleotide sequence represented by SEQ ID NO:2;or

(d) DNA encoding a protein having an endonuclease activity forrecognizing the nucleotide sequence: GCCCAGACATATCCCTGAATGATACC, the DNAbeing capable of hybridizing with DNA which comprises the nucleotidesequence represented by SEQ ID NO:2 under stringent conditions.

Furthermore, the present invention relates to a recombinant vectorcomprising the above-described gene.

Additionally, the present invention relates to a transformant comprisingthe above-described recombinant vector.

Moreover, the present invention relates to a process for producing theendonuclease, comprising the steps of:

culturing the above-described transformant; and

recovering from the culture an endonuclease capable of recognizing thenucleotide sequence: GCCCAGACATATCCCTGAATGATACC.

This specification includes part or all of the contents as disclosed inthe specification and/or drawings of Japanese Patent Application No.10-141861 which is a priority document of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amino acid sequences of an endonuclease before and afterthe modification;

FIG. 2 shows the steps for constructing plasmid pY673L;

FIG. 3 shows the steps for constructing plasmids pEN1.7 and pEN0.5;

FIG. 4 shows the nucleotide sequence of 50 kDa subunit gene of SceIwhich has been modified to conform the universal code;

FIGS. 5A and 5B are photographs of electrophoresis showingsequence-specific endonuclease activities of the 50 kDa subunit of themodified SceI;

FIGS. 6A and 6B show substitution site of the 50 kDa subunit fromSaccharomyces uvarum and oligonucleotides used for the substitution; and

FIGS. 7A and 7B are photographs of electrophoresis showingsequence-specific endonuclease activities of the 50 kDa subunit fromSaccharomyces uvarum.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention will be described in more detail.

The present invention aims at mass-expressing mitochondrial genome DNAencoding the smaller subunit (50 kDa) of an endonuclease from yeast byusing a protein expression system such as E.coli or yeast. Inaccomplishing this aim, the present invention modifies, in a gene codingfor an amino acid sequence of the smaller subunit, codons unique tomitochondria into universal codons. The present invention relates tosuch a modified smaller subunit capable of recognizing and cleaving 26base pairs of the specific nucleotide sequence.

An endonuclease of the present invention (i.e., the 50 kDa subunit of anendonuclease from yeast; hereinafter also referred to as “Endo.SceI 50kDa”) is prepared as follows.

(1) Designing mutated amino acid and introducing mutation

According to the present invention, the smaller subunit of endonucleaseScei from Saccharomyces cerevisiae or the smaller subunit ofendonuclease SuvI from Saccharomyces uvarum is used as a target forintroducing a mutation. The smaller subunits of both SceI and SuvI havemolecular weights of 50 kDa. However, they differ from each other forhaving 2 different amino acids (FIG. 6A).

The gene coding for the subunit (50 kDa) of SceI (hereinafter, referredto as “ENS2”) is encoded by a mitochondrial genome, and thus containsgenetic codes unique to mitochondria (Table 1).

TABLE 1 Difference between mitochondrial code and universal code Aminoacid to be translated Codon Universal code Mitochondrial code TGA STOPTrp ATA Ile Met CTA or CTT Leu Thr

The nucleotide sequence of ENS2 is known (JP-B-7-77556; Nakagawa, K.,Morishima, N., and Shibata, T., J. Biol. Chem. 266, 1977-1984 (1991)).When ENS2 is expressed in a general expression system such as E.coliaccording to the universal code, the translation is interrupted at TGAwhere it is read as a stop codon as can be appreciated from Table 1 (forexample, ENS2 described in JP-B-7-77556 includes a stop codon TGA atnucleotides 97-99). While ATA is read as Ile according to the universalcode, it is read as Met according to the mitochondrial code.

In order to construct a normal mass-expression system for ENS2, it isnecessary to modify the genetic code of ENS2 such that the amino acidsequence obtained upon expression in a general expression system (e.g.,E.coli) is identical to an amino acid sequence as expressed in amitochondrial expression system. Thus, according to the presentinvention, a codon for Trp (TGA) according to the mitochondrial code(Table 1) is substituted for codon (TGG) that will be translated intoTrp according to the universal code. Such substitution is also appliedto ATA, and CTA and CTT that are translated into Ile and Leu,respectively, according to the universal code (Table 1). There is noneed of substituting other degenerating codons which code for Ile or Leuaccording to the universal code.

Basically, there are 37 amino acids as the candidates for modificationwithin the amino acid sequence of the smaller subunit of SceI (476 aminoacids). Their positions are shown in FIG. 1 and Table 2. As to theendonuclease from Saccharomyces uvarum (SuvI), Gly at 217 and Asn at 346(FIG. 1) are additionally substituted for Lys and Asp, respectively, sothat a total of 39 amino acids are modified. It is not necessary tosubstitute all of the above 37 or 39 amino acids. The number ofsubstitution may be 36, or 35 or less. Translations into codes unique tomitochondria may not be complete as long as the 26 nucleotides (SEQ IDNO:1) mentioned later are recognized by the endonuclease. The positionsof substitution are summarized in Table 2 below.

TABLE 2 Amino acid to be Amino acid to be Position of translated beforetranslated after substitution modification modification 33, 54, 247,320, 433 STOP Trp 35, 40, 45, 48, 65, 80, 86, 92, 107, 109, Ile Met 111,123, 154, 163, 168, 171, 177, 248, 313, 335, 347, 399, 465 49, 99, 130,135, 222, Leu Thr 267, 276, 395, 426

Substitution of the amino acids is conducted by substituting thenucleotide sequence of the gene encoding the amino acids for anothernucleotide sequence (site-directed mutagenesis). Examples of mutagenesisinclude but not limited to the site-directed mutagenesis method by T.Kunkel (Kunkel, T. A., Proc. Natl. Acad. Sci. U.S.A. 82, 488-492 (1985))and the Gapped duplex method. There is also a modified version of Kunkelmethod in which a maximum of 16 oligonucleotides for modification aresimultaneously used (instead of using 1 or 2 oligonucleotides as theusual Kunkel method) to efficiently substitute a plurality of sites.According to the present invention, mutation can be introduced by usinga mutation introduction kit (for example, Mutant-K (Takara Shuzo, Co.,Ltd.) or Mutant-G (Takara Shuzo, Co., Ltd.)) that utilizes site-directedmutagenesis, or by using LA PCR in vitro Mutagenesis series kit (TakaraShuzo, Co., Ltd.).

The oligonucleotides are designed and synthesized using the nucleotidesequence of ENS2 as a template (1431 base pairs: Nakagawa, K. et al., J.Biol. Chem. 266, 1977-1984 (1991); JP-B-7-77556) such that at least onebase that is to be introduced with the mutation is flanked by about 8 to30 bases (each oligonucleotide having a total of about 18 to 60 bases).The oligonucleotides can be obtained through chemical synthesis using ausual synthesizer.

(2) Preparation of endonuclease gene which has been introduced withmutation

Each of the oligonucleotides obtained as described in (1) above isphosphorylated at 5′ end, synthesized using ENS2 as a template andsubjected to ligation reactions. These reactions can be performed usingT4 Polynucleotide Kinase (Takara Shuzo, Co., Ltd.), T4 DNA polymerase(Takara Shuzo, Co., Ltd.), T4 DNA ligase (Takara Shuzo, Co., Ltd.) orthe like.

The nucleotide sequence of the thus-obtained DNA is determined. Thedetermination of the nucleotide sequence may be conducted according to aknown method such as Maxam-Gilbert chemical modification method or adideoxynucleotide chain termination method using M13 phage. Generally,the sequence is determined by using an automatic nucleotide sequencer(e.g., ALF (Pharmacia), 373A DNA sequencer (Perkin-Elmer), etc.).

SEQ ID NOS:2 and 3 exemplify the nucleotide sequence of the gene of thepresent invention and the amino acid sequence of the endonuclease of thepresent invention, respectively. The endonuclease of the inventionacquires the essential function of endonuclease SceI or SuvI, i.e., thefunction of recognizing the consensus sequence “CANRYNNANNCYYGTTW” and asequence similar thereto, by linking to the larger subunit of naturalendonuclease. The endonuclease of the invention exerts the function ofthe smaller subunit of the natural endonuclease and can specificallyrecognize the 26 bases represented by “GCCCAGACATATCCCTGAATGATACC” (SEQID NO:1).

The endonuclease of the present invention may include at least onemutation such as deletion, substitution, addition or the like of theamino acid as long as it can specifically recognize the above 26 bases(SEQ ID NO:1).

For example, the amino acid sequence represented by SEQ ID NO:3 mayinclude deletion of at least one, preferably 1 to 10, more preferably 1to 5 amino acids; addition of at least 1, preferably 1 to 10, morepreferably 1 to 5 amino acids; or substitution of at least 1, preferably1 to 10, more preferably 1 to 5 amino acids for another amino acids. Theendonuclease of the invention does not have to include mutations of allof the above-described 37 or 39 amino acids as long as it can recognizethe above 26 bases (SEQ ID NO:1).

The phrase “can recognize” as used herein refers to the function of theendonuclease of the invention to bind to a site of the 26 bases withinthe gene and to cleave the gene such that the 26 base pairs areseparated into two fragments with staggered ends.

DNA that can hybridize with the above gene (SEQ ID NO:2) under stringentconditions may also be included in the gene of the present invention.The stringent conditions are, for example, a sodium concentration of 15to 900 mM and a temperature of 37 to 70° C., preferably 68° C.

(3) Preparation and transformation of recombinant vector

(i) Preparation of recombinant vector

A recombinant vector of the invention may be obtained by ligating(inserting) the gene of the invention to (into) a suitable vector. Thevector for inserting the gene of the invention is not limited to aspecific one as long as it is replicable in a host cell. Examples ofsuch vector include but not limited to plasmid DNA and phage DNA.

The plasmid DNA is, for example, plasmid from E.coli (e.g., pRSET,pTZ19R, pBR322, pBR325, pUC118, pUC119, etc.), plasmid from bacillus(e.g., pUB110, pTP5, etc.), or plasmid from yeast (e.g., YEp13, YEp24,YCp50, etc.). The phage DNA is, for example, λ phage or the like.Similarly, an animal virus vector such as a retrovirus or vaccinia virusvector, or an insect virus vector such as a baculovirus vector may alsobe used.

In order to insert the gene of the invention into the vector, thepurified DNA is cleaved with a suitable restriction enzyme. Then, thecleaved fragment is inserted into the restriction site or a multicloningsite of the suitable vector DNA.

The gene of the present invention should be integrated into the vectorsuch that the gene is able to function. If desired, the vector of theinvention may include, other than the gene of the invention and thepromoter, for example, a cis-element (e.g., an enhancer), a splicingsignal, a poly(A) tail signal, a selective marker, and a ribosomebinding sequence (SD sequence). Examples of the selective marker includedihydrofolate reductase gene, ampicillin-resistant gene andneomycin-resistant gene.

(ii) Preparation of transformant

A transformant of the invention may be obtained by introducing therecombinant vector of the invention into a host cell in such a mannerthat the gene of interest is capable to be expressed. The host cell isnot limited to a specific one as long as it can express the DNA of thepresent invention. Bacteria such as genus Escherichia (e.g., Escherichiacoli), genus Bacillus (e.g., Bacillus subtilis), genus Pseudomonal(e.g,. Pseudomonas putida), genus Rhizobium (e.g., Rhizobium meliloti),yeast such as Schizosaccharomyces pombe, animal cells (e.g., COS and CHOcells), and insect cells (e.g., Sf9 and Sf21) are exemplified.

When a bacterium such as E.coli is used as the host, it is preferablethat the recombinant vector of the present invention is capable ofautonomous replication and includes a promoter, a ribosome bindingsequence, the gene of the invention and a transcription terminationsequence. The recombinant vector may also include a gene for controllingthe promoter.

As the E.coli, E. coli K12 and DH1 are exemplified and as bacillus,Bacillus subtilis MI 114 and 207-21 are exemplified.

As the promoter, any promoter may be used as long as it can be expressedin the host cell like E.coli. For example, a promoter derived fromE.coli or a phage, e.g., trp promoter, lac promoter, p_(L) promoter orp_(R) promoter, may be used. Artificially designed and modified promoterlike tac promoter may also be used.

The recombinant vector may be introduced into the bacterium according toany method for introducing DNA into a bacterium. For example, calciumion method (Cohen, S. N. et al., Proc. Natl. Acad. Sci., USA, 69:2110-2114 (1972)) and an electroporation method may be employed.

An yeast such as Saccharomyces cerevisiae, Saccharomyces uvarum,Schizosaccharomyces pombe or Pichia pastoris may also be used as thehost. In this case, the promoter may be any promoter that can beexpressed in the yeast. Examples of such promoter include but notlimited to gal1 promoter, gal10 promoter, heat shock protein promoter,MF 1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoterand AOX1 promoter.

The recombinant vector may be introduced into the yeast by any methodfor introducing DNA into an yeast. For example, electroporation method(Becker, D. M. et al., Methods Enzymol., 194, 182-187 (1990)),spheroplast method (Hinnen, A. et al., Proc. Natl. Acad. Sci., USA, 75,1929-1933 (1978)), or lithium acetate method (Itoh, H., J. Bacteriol.,153, 163-168 (1983)) may be employed.

An animal cell such as simian cell COS-7, Vero, Chinese hamster ovarycell (CHO cell), mouse L cell, rat GH3 or human FL cell may also be usedas the host. As a promoter, for example, SR promoter, SV40 promoter, LTRpromoter or CMV promoter may be used. Other than these promoters, anearly gene promoter of human cytomegalovirus may also be used.

The recombinant vector may be introduced into the animal cell, forexample, by an electroporation method, a calcium phosphate method or alipofection method.

An insect cell such as Sf9 cell, Sf21 cell or the like may also be usedas the host. The recombinant vector may be introduced into the insectcell, for example, by a calcium phosphate method, a lipofection methodor an electroporation method.

(5) Production of endonuclease

The endonuclease of the present invention may be obtained by culturingthe above-described transformant, and recovering the endonuclease fromthe culture thereof. The term “culture” as used herein refers to aculture supernatant, a cultured cell or microbial cell, or a cell ormicrobial cell debris.

The transformant of the invention is cultured according to a generalmethod used for culturing the host.

A medium for culturing the transformant obtained from a microorganismhost such as E.coli or yeast may be either a natural or a syntheticmedium as long as it contains carbon sources, nitrogen sources,inorganic salts and the like assimilable by the microorganism, and aslong as it can efficiently culture the transformant.

As carbon sources, carbohydrate such as glucose, fructose, sucrose,starch; organic acids such as acetic acid, propionic acid; and alcoholssuch as ethanol and propanol may be used.

As nitrogen sources, ammonia; ammonium salts of inorganic or organicacids such as ammonium chloride, ammonium sulfate, ammonium acetate,ammonium phosphate; other nitrogen-containing compounds; Peptone; meatextract; corn steep liquor and the like may be used.

As inorganic substances, potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, magnesium phosphate, magnesium sulfate, sodiumchloride, iron(II) sulfate, manganese sulfate, copper sulfate, calciumcarbonate and the like may be used.

The cultivation is generally performed under aerobic conditions such asshaking or aeration agitating conditions at 37° C. for 12 to 18 hours.During the cultivation, pH is maintained at 6.5 to 7.5, preferably 7.0.pH is regulated with an inorganic or organic acid, an alkali solution orthe like.

During the cultivation, an antibiotic such as ampicillin, tetracyclineor the like may be added to the medium if necessary.

When culturing a microorganism transformed with an expression vectorusing an inducible promoter, an inducer may be added to the medium atneed. For example, when a microorganism transformed with an expressionvector using Lac promoter or trp promoter is cultured, isopropyl1-thio-β-D-galactoside (IPTG) or indoleacetic acid (IAA) may be added tothe medium, respectively.

A transformant obtained by using an animal cell host may be cultured ina generally used medium such as RPMI1640 medium or DMEM medium, or amedium obtained by supplementing the generally used medium with fetalbovine serum and the like.

The cultivation is generally conducted under 5% CO₂ at 37° C. for 1 to 3days. During the cultivation, an antibiotic such as kanamycin,penicillin or the like may be added to the medium.

After the cultivation, where a microbial cell or a cell intracelluralyproduced endonuclease of the invention, the endonuclease is extracted bydisrupting the microbial cell or the cell. Where a microbial cell or acell extracellularly produced endonuclease of the invention, the culturesolution is directly used. Alternatively, the microbial cell or the cellis removed through centrifugation or the like before isolating andpurifying the endonuclease of the invention from the culture through ageneral biochemical method for protein isolation and purification suchas ammonium sulfate precipitation, gel chromatography, ion exchangechromatography, affinity chromatography, or a combination thereof.

EXAMPLES

Hereinafter, the present invention will be described in detail by way ofexamples which do not limit the technical scope of the presentinvention.

Example 1

Preparation of Single-stranded Template DNA Encoding Subunit of SceI andContaining Deoxyuracil

Endo.SceI 50 kDa subunit gene ENS2 (1431 base pairs; Nakagawa, K.,Morishima, N., and Shibata, T., J. Biol. Chem. 266, 1977-1984 (1991))was modified simultaneously within two regions of the gene, i.e., withinthe upstream moiety of 1.0 kilobase pair and the downstream moiety of0.4 kilobase pair. For this purpose, EcoRI/EcoRI fragment (1671 basepairs) containing the full-length 50 kDa subunit gene (Nakagawa, K.,Morishima, N., and Shibata, T. J., Biol. Chem. 266, 1977-1984 (1991)),and PstI/EcoRI fragment (534 base pairs) containing the downstreammoiety of the 50 kDa subunit gene were separately cloned into phagemidspUC118 (Takara Shuzo, Co., Ltd.), and were named plasmids pEN1.7 andpEN0.5, respectively (FIG. 3). These phagemids were introduced intoE.coli strains CJ236 (Takara Shuzo, Co., Ltd.) for transformation. Thetransformant E.coli strains were shake cultured at 37° C. for 12 hoursor longer so as to prepare pre-culture solutions. Twenty μl of eachpre-culture solution was added to 2 ml of 2×YT culture medium containingampicillin (100 μg/ml) (Sambrook, J., Fritsch, E. F., and Maniatis, T.,Molecular Cloning: a laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) and culturedat 37° C. for 1 hour. To each medium, helper phage M13KO7 (2.0×10¹²plaque-forming unit (pfu); Takara Shuzo, Co., Ltd.) was added toconstitute 0.4% in volume of the medium, and the resultant was culturedat 37° C. for 1 hour. Thereafter, kanamycin (100 μg/ml) was added andthe resultant was cultured at 37° C. for 14 hours. Phage particlesreleased from E.coli into the media during the cultivation wererecovered. Specifically, 1.5 ml of each culture solution was centrifuged(14,000 rpm, 5 min.) in a micro-centrifuge. 1.2 ml of the supernatantwas collected and centrifuged under the same conditions to completelyremove the cell, thereby obtaining 1.0 ml of the supernatant. Subsequentprocedure for preparing the DNA was conducted according to DNApurification of bacteriophage M13 phage summarized in the experimentaltext of J. Sambrook et al. (Sambrook, J., Fritsch, E. F., and Maniatis,T. Molecular Cloning: a laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)).

Example 2

Synthesis of Oligonucleotides for Introducing Site-directed Mutation

(i) Synthesis of single strand

50 kDa subunit gene ENS2 is encoded by mitochondria genome, and thusincludes genetic code unique to mitochondria (Table 1). In order tosubject ENS2 to a general mass-expression system, these unique codonsmust be replaced so as to correspond to the universal code. According toExample 2, the bases were substituted by using a modified method of T.Kunkel (Kunkel, T. A., Proc. Natl. Acad. Sci. U.S.A. 82, 488-492(1985)). Whereas the general Kunkel method uses only one or twooligonucleotides for modification, the present method simultaneouslyused a maximum of 16 oligonucleotides for efficient substitution atmultiple sites.

33 oligonucleotides were designed which each contained the base to besubstituted flanked by approximately 10 to 15 bases (Table 3).

TABLE 3 1. AAAAGACTGGATTATAGAA (SEQ ID NO:6) (A) 2. TGAATATATGTATAAATTT(SEQ ID NO:7) (A) 3. TATTAAATGGGATAATAAT (SEQ ID NO:8) (A) 4.TATTAGATATGTATTATAATG (SEQ ID NO:9) (A) 5. TACACCTATGTCTAATAAA (SEQ IDNO:1O) (A) 6. AAAATATTATGGATTATAAA (SEQ ID NO:11) (A) 7.TTTTATATTTTAAATAAAATGAAAATGGAAATGGATAATTATAATAATAATA (A) (A) (A) (SEQ IDNO:12) 8. AAAATATTATGAATAATTTAA (SEQ ID NO:13) (A) 9.ACTATCTAATATTGAAACTAATTTATCTAATAATTT (SEQ ID NO:14) (CT) 10.TTATTTAATGGATAAATAT (SEQ ID NO:15) (A) 11. ATAAATATATGAAATATTTAG (SEQ IDNO:16) (A) 12. ATAATTATATGTTTAATAATA (SEQ ID NO:17) (A) 13.GGAGGTATTACAATTACTAATCATGCTAATGAT (SEQ ID NO:18) (CTA) 14.TTTTAGTAGAAAAATGGATGGATACTTTAAAAGATA (SEQ ID NO:19) (A) (A) 15.AGCTAAAGAAAAGATTTTTACTAATATTTATAATAATTA (SEQ ID NO:20) (CT) 16.AAATATTATGGATATTAAA (SEQ ID NO:21) (C) 17. TAATTATTGGTTATCTGG (SEQ IDNO:22) (A) 18. ATCATCTATGTATAATCCT (SEQ ID NO:23) (A) 19.TTAAAAATATGAGACCTAG (SEQ ID NO:24) (A) 20. GATGAATTAATGAAATTTATTTA (SEQID NO:25) (A) 21. ATTAAATTTAGATTTAATACTTTTATTAAATCATATAAT (SEQ ID NO:26)(CTA) 22. TATAATAAATATATTAATATGCATAATGCACGTAAACC (SEQ ID NO:27) (A) 23.TAAATTTTTAATAAATAATATGACTTGTTTTATTAAATGgGA (SEQ ID NO:28) (ACTA) 24.AAGATTAATGAATTCAAAA (SEQ ID NO:29) (A) 25.GATTATAAATTATTATATACTTATTTTTATATTTTAAAT (SEQ ID NO:30) (CT) 26.gAATAATTTAAATTATAAAACTTCTAATATTGAAacTA (SEQ ID NO:31) (CTA) 27.TTCTCTATTAATATTAAAACTAATTTAGCTAAAGAAA (SEQ ID NO:32) (CT) 28.AAATTATTTACCAGAACTACTGATGAATTAATgAAATT (SEQ ID NO:33) (CT) 29.CATATAATTGGAATAATAGA (SEQ ID NO:34) (A) 30. AATTTTTAATGAATAATATg (SEQ IDNO:35) (A) 31. TTTAGATATGTTAAATATg (SEQ ID NO:36) (A) 32.ATATgTTAAATATGATTCCTAATAA (SEQ ID NO:37) (A) 33. CTGgATTATGGAATATGAAT(SEQ ID NO:38) (A)

In Table 3, the base(s) in parentheses underneath each sequencerepresent the original base(s) that was (were) substituted for theunderlined base(s). The bases shown in small letters represent thosewhich have already replaced the original oligonucleotide.

The lengths of the oligonucleotides vary within the range of 18 to 52bases and they include mutation of 1 to a maximum of 4 residues. Theseoligonucleotides were used for substituting 50 base pairs of the 1431 bp50 kDa subunit gene to modify 37 codons. 5′ end of each oligonucleotidewas phosphorylated so as to allow the DNA ligase reaction describedlater. The composition of the reaction mixture for the phosphorylationis shown below:

100 mM Tris-HCl (pH 8.0)

10 mM magnesium chloride

7 mM dithiothreitol

1 mM ATP, 1 μM oligonucleotide

T4 polynucleotide kinase (15 units)

Total amount 30 μl

The reaction mixture was subjected to phosphorylation reaction at 37° C.for 15 min., and then the enzyme was inactivated through a treatment at70° C. for 10 min.

(ii) Synthesis of complementary strand

The oligonucleotides obtained in (i) were treated as follows to obtaindouble-strands. Compositions of the annealing buffer and the elongationreaction buffer are shown below:

Annealing Buffer

200 mM Tris-HCl (pH 8.0)

100 mM magnesium chloride

500 mM sodium chloride

10 mM dithiothreitol

Elongation Reaction Buffer

50 mM Tris-HCl (pH 8.0)

5 mM dithiothreitol

60 mM ammonium acetate

0.5 mM each of dNTPs (A, C, T, G)

5 mM magnesium chloride

1 mM nicotinamido adenine dinucleotide

Distilled water was added to 1 μl of the annealing buffer and 0.2 pmolof the single-stranded template DNA, resulting in a total amount of 10μl. One μl of the solution was dispensed to be mixed with 1 μl of thephosphorylated oligonucleotide solution. The resultant mixture was leftto stand at 65° C. for 15 min. and then at 37° C. for 15 min., wherebythe oligonucleotide annealed to the single-stranded DNA. To thesolution, 25 μl of the elongation buffer, 60 units of E.coli DNA ligaseand 1 unit of T4 DNA polymerase were added and left to stand at 25° C.for 2 hours so as to synthesize a complementary strand. Three μl of 0.2M ethylene diamine tetra acetic acid tetrasodium salt (pH 8.0) was addedto terminate the enzyme reaction, after which the enzyme was inactivatedthrough treatment at 65° C. for 5 min. This reaction solution wasdirectly used for the subsequent transformation.

Example 3

Transformation

E.coli BMH71-18 muts (Takara Shuzo, Co., Ltd.) was used such that thenucleotide sequence of the wild-type DNA strand (the single-stranded DNAprepared with CJ236) in the double-stranded plasmid DNA obtained throughthe complementary strand synthesis was substituted for a mutant type. Inthis E.coli strain, deoxyuracil contained in the single-stranded DNAprepared with CJ236 was hydrolyzed by enzyme uracil-DNA glycosylase andthen synthesized again using the DNA strand containing the substitutedbase as a template (Lindahl, T., Ann. Rev. Biochem. 51, 61-87 (1982).

The whole reaction mixture with the synthesized complementary strand wasadded to 100 μl solution containing competent cell of BMH71-18 muts.E.coli competent cell was prepared according to the method of H. Inoueet al. (Inoue, H., Nojima, H., and Okayama, H., Gene 96, 23-28 (1990)).

To the solution, a medium was added and left at 37° C. for 1 hour. Then,30 μl of helper phage (supra) was added and left to stand at 37° C. foranother 30 min. for infection to take place. 40 μl of culture solutionof BMH71-18 intracellularly containing both the helper phage and theplasmid was fractionated and added to 2 ml of 2×YT medium containingampicillin (100 μg/ml) and kanamycin (100 μg/ml). The resultant mediumwas shake cultured at 37° C. for 16 to 20 hours to produce a phage.

The microbial cell was removed through centrifugation (14,000 rpm, 5min.). The supernatant was collected which contained the phage particleincorporating the single-stranded DNA of the plasmid with thesubstituted base. With 20 μl of the supernatant, 80 μl of strain MV1184(Takara Shuzo, Co., Ltd.) which has been cultured for 12 hours or longerwas mixed and left to stand at 37° C. for 10 min. so as to inject thesingle-stranded DNA of the phage into the cell. The strain MV1184containing the plasmid resulting from replication of the integratedsingle-stranded DNA was inoculated to an LB agar medium containing 100μg/ml ampicillin (Sambrook, J., Fritsch, E. F., and Maniatis, T.,Molecular Cloning: a laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor N.Y. (1989)) for selection.The nucleotide sequences of the 50 kDa subunit genes were analyzed forsome clones with an automatic sequencer ALF (Pharmacia) to confirm theincorporation of the predetermined substitutions. The fluorescent primerfor the analysis of the nucleotide sequence was purchased from Pharmacia(Uppsala, Sweden). The DNA sequencing reaction was based on the Sangermethod (Sanger, F. et al., Proc. Natl. Acad. Sci., 74, 5463-5467 (1977))according to the protocol of Pharmacia.

For the starting material, plasmid pEN1.7, forty nucleotidesubstitutions were performed by 9 cycles of the mutagenic process, while10 nucleotide substitutions were performed for pEN0.5 by 4 cycles ofsuch process. Once all of the substitutions were confirmed, the upstreamand downstream moieties were linked at the PstI cleavage site, therebyobtaining a gene of the invention encoding the 50 kDa subunit withcomplete substitutions (FIG. 4, SEQ ID NO:2).

Example 4

Construction of Expression Plasmid for 50 kDa Subunit

For facilitating the linking between the modified 50 kDa subunit geneand the vector for inducing expression thereof, restriction sites wereintroduced into the 5′ and 3′ terminuses of the modified gene throughpolymerase chain reaction (PCR). The reaction was performed using TaqDNA polymerase (Takara Shuzo, Co., Ltd.) according to the protocol ofthe manufacturer. Sequences of the used primers are shown below.

5′-CCGGATCCATGAAAAAAC-3′ (SEQ ID NO:4)

5′-GGGTCGACTTATTTAATGTATCC-3′ (SEQ ID NO:5)

The underlined parts are the newly introduced BamHI and SalI recognitionsequences. The reaction was performed through 25 cycles of: 94° C. for 1min.; 45° C. for 2 min.; and 72° C. for 3 min.

The DNA fragment (1447 base pairs) amplified by PCR was separatedthrough agarose (0.8%) gel electrophoresis (Sambrook, J., Fritsch, E.F., and Maniatis, T. Molecular Cloning: a laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989)) and stained with ethidium bromide for confirmation. The fragmentwas recovered from the agarose gel using Geneclean kit (BIO101,California, USA).

The recovered DNA fragment was treated with BaMHI and SalI, andsubcloned into pRSET (Invitrogen Corp.) and pTZ19R (Pharmacia) to obtainpSC50 and pTZSC50, respectively. Plasmid pSC50 was used for inducing theexpression. Plasmid pTZSC50 was subjected to DNA sequencing usingfluorescent primer (supra) so as to confirm that no extra mutation hadbeen introduced during the PCR.

Example 5

Induction of Expression of 50 kDa Subunit

Expression plasmid pSC50 was introduced into a competent cell of E.coliBL21 (DE3) pLysS (Invitrogen Corp.). The transformant cell waspre-cultured through shake cultivation in an LB liquid medium containingampicillin (150 μg/ml) and chloramphenicol (34 μg/ml) (Sambrook, J.,Fritsch, E. F., and Maniatis, T. Molecular Cloning: a laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989)) at 37° C. overnight. The pre-cultured solution (4% involume of a resultant culture) was centrifuged (2,500×g, 10 min.) torecover the microbial cell. This precipitate was suspended in a smallamount of fresh medium, which was then added to a liquid medium. Shakecultivation (37° C.) was performed until the suspension level at 600nanometers (nm) (OD600) of about 0.5 was obtained. Thereafter, shakecultivation was continued at 18° C. When the OD600 became about 0.8,isopropyl 1-thio-β-D-galactoside (IPTG) was added to the finalconcentration of 0.4 mM to initiate induction of expression of the 50kDa subunit. After performing shake cultivation at 18° C. for another 12hours, E.coli was recovered through centrifugation, rapidly frozen withliquid nitrogen and stored at −80° C.

Example 6

Purification of 50 kDa Subunit from E.coli

The microbial cell stored at −80° C. was melted at room temperature. Thesubsequent treatments were conducted at 4° C. or on ice. The microbialcell was suspended in Buffer A (20 mM Tris-HCl buffer (pH 8.0), 500 mMsodium chloride, 5 mM imidazole, 1 mM phenylmethylsulfonyl fluoride(PMSF; Sigma Aldrich Japan K.K., Tokyo, Japan), 0.1% NP-40 (NacalaiTesque, Inc., Kyoto, Japan)). The resultant suspension was rapidlyfrozen with liquid nitrogen and melted under running water to disruptthe E.coli. The suspension was treated for 5 times with anultrasonicator (UR-200P, Tomy Seiko Co., Ltd., Tokyo, Japan) at amaximum output for 30 sec.

The treated solution was centrifuged (39,000×g, 20 min.) at 4° C. Theobtained supernatant was filtrated through 0.45 μm Mylex filter(Millipore, Mass., USA). The sample was placed in a column (φ10 mm, 2.0ml) loaded with Probond Nickel Chelate Resin (Invitrogen Corp.) whichhad been equibralized with Buffer A. Then, the sample was washed with 20ml of Buffer A (ten times the volume of the resin). After anotherwashing with 12 ml Buffer A containing 60 mM imidazole (six times thevolume of the resin), the resultant was subjected to gradient elutionwith 60-50 mM imidazole-containing Buffer A (total amount of 80 ml). Theeluted fraction was subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis (Laemmli, U. K. Nature, 227, 680-685 (1970)) andstained with Coomasie brilliant blue to confirm the presence of the 50kDa subunit. The fraction containing the 50 kDa subunit was dialyzedagainst Buffer B (20 mM Tris-HCl buffer (pH 7.5), 300 mM sodiumchloride, 1 mM ethylenediaminetetraacetic acid tetrasodium salt, 1 mMdithiothreitol). The purified protein was quantified using Protein assayagent (Bio-Rad, California, USA) according to the micro-assay method ofthe manufacturer. Bovine serum albumin solution (Sigma Aldrich JapanK.K.) was used as a standard protein. As a result, 300 μg of purifiedprotein was obtained from 25 g (wet weight) of the microbial cell.

Example 7

Measurement of Endonuclease Activity

A substrate for measuring an endonuclease activity of the 50 kDa subunitwas prepared as follows. An EcoRI/EcoRI fragment containing oli2 regionon mitochondria DNA within which Endo.SceI is known to be cleaved (1671base pairs; Nakagawa, K. et al., EMBO J. 11, 2707-2715 (1992)) wassubcloned into phagemid pUC119 (Takara Shuzo, Co., Ltd.), the resultantcalled pY673L (FIG. 2). Plasmid pBR322 (Takara Shuzo, Co., Ltd.) wasused as a control DNA substrate. Plasmids pY673L and pBR322 were used totransform E.coli. Then, the plasmids were extracted from E.coli andhighly purified using Qiagen column (Qiagen Japan, Tokyo, Japan).

The composition of the reaction solution used for measuring theendonuclease activity of the 50 kDa subunit is shown below:

50 mM Tris-HCl buffer (pH 8.0)

50 mM sodium chloride

10 mM magnesium chloride

1 mM dithiothreitol

25 ng substrate DNA (pY673L or pBR322 which has been linearized withrestriction enzyme ScaI (FIG. 2))

0.4 to 60 ng 50 kDa subunit

Total volume 30 μl

After performing the DNA cleavage reaction at 37° C. for 30 min.,ethylenediaminetetraacetic acid tetrasodium salt and sodium dodecylsulfate were added to final concentrations of 10 mM and 0.3%,respectively, to terminate the reaction. The cleaved DNA was subjectedto 0.8% agarose electrophoresis either directly or after concentratingthe DNA through phenol extraction and ethanol precipitation.

After the electrophoresis, the gel was stained with ethidium bromide(Sigma Aldrich Japan K.K.) or SYBR Green (Takara Shuzo, Co., Ltd.) toconfirm cleavage of DNA. The DNA was detected using FMBIO Imaging device(Takara Shuzo, Co., Ltd.) to determine the DNA cleavage.

Example 8

Detection of Sequence-specific Endonuclease

The dimeric Endo.SceI recognizes and cleaves in vivo and in vitro 26base pairs similar to the consensus sequence within the oli2 gene regionon the mitochondria DNA (Nakagawa, K., Morishima, N., and Shibata, T.,EMBO J. 11, 2707-2715 (1992)) (FIG. 2). The purified 50 kDa subunit ofEndo.SceI cleaved a specific sequence (SEQ ID NO:1) by itself.

Specific cleavage of oli2 with the 50 kDa subunit using plasmid pY673Lcontaining oli2 as the substrate was confirmed (FIG. 5A). Referring toFIG. 5A, Lanes 1 to 8 are the results obtained with 0.5, 1.0, 2.0, 4.0,8.0, 16.0, 32.0 and 64.0 ng of the 50 kDa subunits, respectively. With64 ng of 50 kDa subunit, 60% of pY673L (25 ng) in the reaction solutionwas sequence-specifically cleaved at 37° C. within 30 min., whereby DNAfragments of 3.4 and 1.4 kilobases were detected.

DNA was not cleaved with the 50 kDa subunit using plasmid pBR322 as thesubstrate and no cleavage fragment was detected (FIG. 5B). Referring toFIG. 5B, Lanes 1 to 7 are the results obtained with 2.3, 4.5, 9.0, 18.0,36.0, 72.0 and 144 ng of the 50 kDa subunits, respectively. When anexcessive amount (200 ng) of the 50 kDa subunit was used, no cleavagewas found with pBR322 or other DNAs (mitochondria DNA (80 kilobasepairs) from bud yeast strain, E.coli phage λ DNA (47 kilobase pairs),and bacillus phage φ105 DNA (38 kilobase pairs) which did not containspecific sequence (26 base pairs) within oli2 gene region).

Example 9

Mass Production of 50 kDa Subunit from Saccharomyces Uvarum andDetection of the Activity Thereof

Endo.SuvI 50 kDa subunit, a homologous protein of Endo.SceI 50 kDasubunit from Saccharomyces cerevisiae is present in Saccharomyces uvarum(Nakagawa, K., Morishima, N., and Shibata, T., J. Bio. Chem. 266,1977-1984 (1991)). Both subunits have 476 amino acid residues but thereare two differences in amino acid level between them. The amino aciddifferences between Endo.SceI and Endo.SuvI 50 kDa subunits are shown inFIG. 6A.

A mass-expression gene for Endo.SuvI 50 kDa subunit was prepared byintroducing two additional modifications into the modified gene forEndo.SceI 50 kDa subunit. The oligonucleotides used for thesubstitutions of the amino acids for Endo.SuvI 50 kDa subunit are shownin FIG. 6B. With reference to FIG. 6B, the bases in parenthesescorrespond to the nucleotide sequence of Endo.SceI 50 kDa subunit. Forthis purpose, two oligonucleotides were newly synthesized to introducemutations according to the gene modification method described above. Themutation was confirmed through DNA sequencing. The modified gene wassubcloned into pRSET vector (Invitrogen Corp.), which was thenintroduced into E.coli BL21 (DE3) pLys by transformation method.

Endo.SuvI 50 kDa subunit was expressed and purified according to themethod applied to Endo.SceI 50 kDa subunit described above. The purifiedEndo.SuvI 50 kDa subunit was used to specifically cleave plasmid pY673L.

As a result, Endo.SuvI 50 kDa subunit was equally effective insequence-specifically cleaving the Endo.SceI 50 kDa subunit cleavagesite on plasmid pY673L (FIG. 7A).

Meanwhile, Endo.SuvI 50 kDa subunit did not cleave plasmid pBR322 at all(FIG. 7B). Also, other DNAs (bud yeast mitochondria DNA, bacillus phageφ105 λ DNA and E.coli phage λ DNA which did not contain oli2 generegion) were not cleaved by Endo.SuvI 50 kDa subunit.

In FIGS. 7A and 7B, Lanes 1 to 7 are the results obtained with 2.3, 4.5,9.0, 18.0, 36.0, 72.0 and 144 ng of 50 kDa subunits, respectively.

According to the present invention, a site-specific endonuclease capableof recognizing a specific nucleotide sequence, a gene encoding theendonuclease, a recombinant vector containing the gene, a transformantcontaining the vector, and a process for producing the endonuclease areprovided. Since the endonuclease of the present invention is capable ofrecognizing a specific sequence of 26 bases, it is useful in the fieldof genetic engineering and biochemistry in modifying and mapping DNA fora wide application, i.e., plasmid to genome.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

The following are information on sequences described herein:

38 1 26 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 1 gcccagacat atccctgaat gatacc 26 2 1431 DNASaccharomyces cerevisiae CDS (1)..(1428) 2 atg aaa aaa caa aat tta aattct att tta tta atg tat att aat tat 48 Met Lys Lys Gln Asn Leu Asn SerIle Leu Leu Met Tyr Ile Asn Tyr 1 5 10 15 att att aat tat ttt aat aatatt cat aaa aat caa tta aaa aaa gac 96 Ile Ile Asn Tyr Phe Asn Asn IleHis Lys Asn Gln Leu Lys Lys Asp 20 25 30 tgg att atg gaa tat gaa tat atgtat aaa ttt tta atg aat aat atg 144 Trp Ile Met Glu Tyr Glu Tyr Met TyrLys Phe Leu Met Asn Asn Met 35 40 45 act tgt ttt att aaa tgg gat aat aataaa att tta tta tta tta gat 192 Thr Cys Phe Ile Lys Trp Asp Asn Asn LysIle Leu Leu Leu Leu Asp 50 55 60 atg tat tat aat gta tta tat aac tat cataaa caa cgt aca cct atg 240 Met Tyr Tyr Asn Val Leu Tyr Asn Tyr His LysGln Arg Thr Pro Met 65 70 75 80 tct aat aaa aga tta atg aat tca aaa aatatt atg gat tat aaa tta 288 Ser Asn Lys Arg Leu Met Asn Ser Lys Asn IleMet Asp Tyr Lys Leu 85 90 95 tta tat act tat ttt tat att tta aat aaa atgaaa atg gaa atg gat 336 Leu Tyr Thr Tyr Phe Tyr Ile Leu Asn Lys Met LysMet Glu Met Asp 100 105 110 aat tat aat aat aat aat aat aat att tca ttaaaa tat aat gaa tta 384 Asn Tyr Asn Asn Asn Asn Asn Asn Ile Ser Leu LysTyr Asn Glu Leu 115 120 125 tta aaa aat att atg aat aat tta aat tat aaaact tct aat att gaa 432 Leu Lys Asn Ile Met Asn Asn Leu Asn Tyr Lys ThrSer Asn Ile Glu 130 135 140 act aat tta tct aat aat ttt tat tta atg gataaa tat tta att aat 480 Thr Asn Leu Ser Asn Asn Phe Tyr Leu Met Asp LysTyr Leu Ile Asn 145 150 155 160 aaa tat atg aaa tat tta gat atg tta aatatg att cct aat aat tat 528 Lys Tyr Met Lys Tyr Leu Asp Met Leu Asn MetIle Pro Asn Asn Tyr 165 170 175 atg ttt aat aat att aat tat aaa ggt aaatta aat att aaa aca gta 576 Met Phe Asn Asn Ile Asn Tyr Lys Gly Lys LeuAsn Ile Lys Thr Val 180 185 190 tta gat tta aat aat aat gaa ttt tat gattat tta tca ggg tta att 624 Leu Asp Leu Asn Asn Asn Glu Phe Tyr Asp TyrLeu Ser Gly Leu Ile 195 200 205 gaa ggt gat ggt tat att ggt cct gga ggtatt aca att act aat cat 672 Glu Gly Asp Gly Tyr Ile Gly Pro Gly Gly IleThr Ile Thr Asn His 210 215 220 gct aat gat gta tta aat act atc ttt attaat aaa aga att aaa aat 720 Ala Asn Asp Val Leu Asn Thr Ile Phe Ile AsnLys Arg Ile Lys Asn 225 230 235 240 agt att tta gta gaa aaa tgg atg gatact tta aaa gat aat cct tat 768 Ser Ile Leu Val Glu Lys Trp Met Asp ThrLeu Lys Asp Asn Pro Tyr 245 250 255 ttt gtt aat gct ttc tct att aat attaaa act aat tta gct aaa gaa 816 Phe Val Asn Ala Phe Ser Ile Asn Ile LysThr Asn Leu Ala Lys Glu 260 265 270 aag att ttt act aat att tat aat aaatta tat agt gat tat aaa att 864 Lys Ile Phe Thr Asn Ile Tyr Asn Lys LeuTyr Ser Asp Tyr Lys Ile 275 280 285 aat caa att aat aat cat atc cct tattat aat tat tta aaa att aat 912 Asn Gln Ile Asn Asn His Ile Pro Tyr TyrAsn Tyr Leu Lys Ile Asn 290 295 300 aat aaa tta cct att aaa aat att atggat att aaa aat aat tat tgg 960 Asn Lys Leu Pro Ile Lys Asn Ile Met AspIle Lys Asn Asn Tyr Trp 305 310 315 320 tta gct ggt ttt aca gct gca gatggt tct ttt tta tca tct atg tat 1008 Leu Ala Gly Phe Thr Ala Ala Asp GlySer Phe Leu Ser Ser Met Tyr 325 330 335 aat cct aaa gat aca tta tta tttaaa aat atg aga cct agt tat gtt 1056 Asn Pro Lys Asp Thr Leu Leu Phe LysAsn Met Arg Pro Ser Tyr Val 340 345 350 att tca caa gtt gaa aca cgt aaagaa tta att tat tta att caa gaa 1104 Ile Ser Gln Val Glu Thr Arg Lys GluLeu Ile Tyr Leu Ile Gln Glu 355 360 365 tct ttt gat tta tct att tct aatgtt aaa aaa gtt ggt aat aga aaa 1152 Ser Phe Asp Leu Ser Ile Ser Asn ValLys Lys Val Gly Asn Arg Lys 370 375 380 tta aaa gat ttt aaa tta ttt accaga act act gat gaa tta atg aaa 1200 Leu Lys Asp Phe Lys Leu Phe Thr ArgThr Thr Asp Glu Leu Met Lys 385 390 395 400 ttt att tat tat ttt gat aaattt tta cct tta cat gat aat aaa caa 1248 Phe Ile Tyr Tyr Phe Asp Lys PheLeu Pro Leu His Asp Asn Lys Gln 405 410 415 ttt aat tat att aaa ttt agattt aat act ttt att aaa tca tat aat 1296 Phe Asn Tyr Ile Lys Phe Arg PheAsn Thr Phe Ile Lys Ser Tyr Asn 420 425 430 tgg aat aat aga gta ttt ggttta gta tta tct gaa tat atc aat aat 1344 Trp Asn Asn Arg Val Phe Gly LeuVal Leu Ser Glu Tyr Ile Asn Asn 435 440 445 att aaa att gat aat tat gattat tat tat tat aat aaa tat att aat 1392 Ile Lys Ile Asp Asn Tyr Asp TyrTyr Tyr Tyr Asn Lys Tyr Ile Asn 450 455 460 atg cat aat gca cgt aaa cctaaa gga tac att aaa taa 1431 Met His Asn Ala Arg Lys Pro Lys Gly Tyr IleLys 465 470 475 3 476 PRT Saccharomyces cerevisiae 3 Met Lys Lys Gln AsnLeu Asn Ser Ile Leu Leu Met Tyr Ile Asn Tyr 1 5 10 15 Ile Ile Asn TyrPhe Asn Asn Ile His Lys Asn Gln Leu Lys Lys Asp 20 25 30 Trp Ile Met GluTyr Glu Tyr Met Tyr Lys Phe Leu Met Asn Asn Met 35 40 45 Thr Cys Phe IleLys Trp Asp Asn Asn Lys Ile Leu Leu Leu Leu Asp 50 55 60 Met Tyr Tyr AsnVal Leu Tyr Asn Tyr His Lys Gln Arg Thr Pro Met 65 70 75 80 Ser Asn LysArg Leu Met Asn Ser Lys Asn Ile Met Asp Tyr Lys Leu 85 90 95 Leu Tyr ThrTyr Phe Tyr Ile Leu Asn Lys Met Lys Met Glu Met Asp 100 105 110 Asn TyrAsn Asn Asn Asn Asn Asn Ile Ser Leu Lys Tyr Asn Glu Leu 115 120 125 LeuLys Asn Ile Met Asn Asn Leu Asn Tyr Lys Thr Ser Asn Ile Glu 130 135 140Thr Asn Leu Ser Asn Asn Phe Tyr Leu Met Asp Lys Tyr Leu Ile Asn 145 150155 160 Lys Tyr Met Lys Tyr Leu Asp Met Leu Asn Met Ile Pro Asn Asn Tyr165 170 175 Met Phe Asn Asn Ile Asn Tyr Lys Gly Lys Leu Asn Ile Lys ThrVal 180 185 190 Leu Asp Leu Asn Asn Asn Glu Phe Tyr Asp Tyr Leu Ser GlyLeu Ile 195 200 205 Glu Gly Asp Gly Tyr Ile Gly Pro Gly Gly Ile Thr IleThr Asn His 210 215 220 Ala Asn Asp Val Leu Asn Thr Ile Phe Ile Asn LysArg Ile Lys Asn 225 230 235 240 Ser Ile Leu Val Glu Lys Trp Met Asp ThrLeu Lys Asp Asn Pro Tyr 245 250 255 Phe Val Asn Ala Phe Ser Ile Asn IleLys Thr Asn Leu Ala Lys Glu 260 265 270 Lys Ile Phe Thr Asn Ile Tyr AsnLys Leu Tyr Ser Asp Tyr Lys Ile 275 280 285 Asn Gln Ile Asn Asn His IlePro Tyr Tyr Asn Tyr Leu Lys Ile Asn 290 295 300 Asn Lys Leu Pro Ile LysAsn Ile Met Asp Ile Lys Asn Asn Tyr Trp 305 310 315 320 Leu Ala Gly PheThr Ala Ala Asp Gly Ser Phe Leu Ser Ser Met Tyr 325 330 335 Asn Pro LysAsp Thr Leu Leu Phe Lys Asn Met Arg Pro Ser Tyr Val 340 345 350 Ile SerGln Val Glu Thr Arg Lys Glu Leu Ile Tyr Leu Ile Gln Glu 355 360 365 SerPhe Asp Leu Ser Ile Ser Asn Val Lys Lys Val Gly Asn Arg Lys 370 375 380Leu Lys Asp Phe Lys Leu Phe Thr Arg Thr Thr Asp Glu Leu Met Lys 385 390395 400 Phe Ile Tyr Tyr Phe Asp Lys Phe Leu Pro Leu His Asp Asn Lys Gln405 410 415 Phe Asn Tyr Ile Lys Phe Arg Phe Asn Thr Phe Ile Lys Ser TyrAsn 420 425 430 Trp Asn Asn Arg Val Phe Gly Leu Val Leu Ser Glu Tyr IleAsn Asn 435 440 445 Ile Lys Ile Asp Asn Tyr Asp Tyr Tyr Tyr Tyr Asn LysTyr Ile Asn 450 455 460 Met His Asn Ala Arg Lys Pro Lys Gly Tyr Ile Lys465 470 475 4 18 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 4 ccggatccat gaaaaaac 18 5 23 DNA ArtificialSequence Description of Artificial SequenceSynthetic DNA 5 gggtcgacttatttaatgta tcc 23 6 19 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 6 aaaagactgg attatagaa 19 7 19 DNA ArtificialSequence Description of Artificial SequenceSynthetic DNA 7 tgaatatatgtataaattt 19 8 19 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 8 tattaaatgg gataataat 19 9 21 DNA ArtificialSequence Description of Artificial SequenceSynthetic DNA 9 tattagatatgtattataat g 21 10 19 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 10 tacacctatg tctaataaa 19 11 20 DNA ArtificialSequence Description of Artificial SequenceSynthetic DNA 11 aaaatattatggattataaa 20 12 52 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 12 ttttatattt taaataaaat gaaaatggaa atggataattataataataa ta 52 13 21 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 13 aaaatattat gaataattta a 21 14 36 DNA ArtificialSequence Description of Artificial SequenceSynthetic DNA 14 actatctaatattgaaacta atttatctaa taattt 36 15 19 DNA Artificial SequenceDescription of Artificial SequenceSynthetic DNA 15 ttatttaatg gataaatat19 16 21 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 16 ataaatatat gaaatattta g 21 17 21 DNA ArtificialSequence Description of Artificial SequenceSynthetic DNA 17 ataattatatgtttaataat a 21 18 33 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 18 ggaggtatta caattactaa tcatgctaat gat 33 19 36DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA19 ttttagtaga aaaatggatg gatactttaa aagata 36 20 39 DNA ArtificialSequence Description of Artificial SequenceSynthetic DNA 20 agctaaagaaaagattttta ctaatattta taataatta 39 21 19 DNA Artificial SequenceDescription of Artificial SequenceSynthetic DNA 21 aaatattatg gatattaaa19 22 18 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 22 taattattgg ttatctgg 18 23 19 DNA ArtificialSequence Description of Artificial SequenceSynthetic DNA 23 atcatctatgtataatcct 19 24 19 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 24 ttaaaaatat gagacctag 19 25 23 DNA ArtificialSequence Description of Artificial SequenceSynthetic DNA 25 gatgaattaatgaaatttat tta 23 26 39 DNA Artificial Sequence Description ofArtificial SequenceSynthetic DNA 26 attaaattta gatttaatac ttttattaaatcatataat 39 27 38 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 27 tataataaat atattaatat gcataatgca cgtaaacc 38 2842 DNA Artificial Sequence Description of Artificial SequenceSyntheticDNA 28 taaattttta ataaataata tgacttgttt tattaaatgg ga 42 29 19 DNAArtificial Sequence Description of Artificial SequenceSynthetic DNA 29aagattaatg aattcaaaa 19 30 39 DNA Artificial Sequence Description ofArtificial SequenceSynthetic DNA 30 gattataaat tattatatac ttatttttatattttaaat 39 31 38 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 31 gaataattta aattataaaa cttctaatat tgaaacta 38 3237 DNA Artificial Sequence Description of Artificial SequenceSyntheticDNA 32 ttctctatta atattaaaac taatttagct aaagaaa 37 33 38 DNA ArtificialSequence Description of Artificial SequenceSynthetic DNA 33 aaattatttaccagaactac tgatgaatta atgaaatt 38 34 20 DNA Artificial SequenceDescription of Artificial SequenceSynthetic DNA 34 catataattg gaataataga20 35 20 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 35 aatttttaat gaataatatg 20 36 19 DNA ArtificialSequence Description of Artificial SequenceSynthetic DNA 36 tttagatatgttaaatatg 19 37 25 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA 37 atatgttaaa tatgattcct aataa 25 38 20 DNAArtificial Sequence Description of Artificial SequenceSynthetic DNA 38ctggattatg gaatatgaat 20

What is claimed is:
 1. A method of cleaving a DNA having a 26 base sequence GCCCAGACATATCCCTGAATGATACC (SEQ ID NO:1), comprising (a) reacting the DNA with a protein comprising the amino acid sequence of SEQ ID NO:3; and (b) cleaving the DNA such that said DNA is cleaved within said 26 base region producing staggered ends.
 2. A method of detecting DNA having a 26 base sequence GCCCAGACATATCCCTGAATGATACC (SEQ ID NO:1), comprising (a) reacting the DNA with a protein comprising the amino acid sequence of SEQ ID NO:3; (b) cleaving the DNA such that said DNA is cleaved within said 26 base region producing staggered ends; and (c) detecting the cleaved DNA. 